Translational studies are proposed to support the continued development of the JAA-F11 antibody based therapy to address the critical need for a safe and effective treatment for metastatic breast cancer, including triple negative breast cancers. Building on the success of our Phase I project, the overarching objectives of the Phase II application are to advance translational studies with humanized JAA-F11 (hJAA- F11). These novel IgG1 antibodies binds with high specificity to the pancarcinoma Thomsen-Friedenreich glycoantigen (TF-Ag; Gal1-3GalNAc alpha), which is a unique target expressed on the surface of about 80% of breast, colon, bladder, prostate and other carcinomas, but not on normal tissues. The successful generation of 3 humanized and one chimeric JAA-F11 constructs, which have the same unique chemical specificity for TF- Ag as our patented mouse monoclonal antibody mJAA-F11 directly supports the hypothesis of our Phase I application, that humanization of JAA-F11 can yield an antibody of the same chemical and biologic specificity as the original mJAA-F11. In addition, there is active progress on generation of up to an additional 6 humanized constructs that will be evaluated to identify lead antibody candidates. The hypothesis of the Phase II application is that humanized constructs (hJAA-F11) can be used therapeutically to create a survival advantage for patients with breast cancers, including triple negative breast cancers, through direct killing and by blocking tumor cell spread. Specific aims of this application are focused on identifying lead drug candidates and generating preclinical data in support of an Investigational New Drug (IND) application for therapeutic and diagnostic applications of hJAA-F11 in breast cancer patients. Aim 1 will identify 2 lead antibody candidates (hJAA-F11 constructs), one of which supports the use of the antibody as a direct cancer immunotherapeutic agent and the second which supports the use as an antibody-drug conjugate. Both candidates will demonstrate high chemical and biological specificity for TF-Ag positive tumors. The lead immunotherapeutic candidate will have high antibody directed cellular cytotoxicity (ADCC) activity and the lead candidate for an antibody drug conjugate will have rapid tumor cell internalization. Aim 2 will assess the efficacy of hJAA-F11 constructs on tumor growth rate in a xenograft human breast cancer model in Nude mice. Efficacy studies will be conducted with the construct having optimal ADCC activity and the construct with optimal internalization will be tested as an antibody-linker-maytansine derivative. Aim 3 will conduct pharmacokinetic studies of these hJAA-F11 constructs in mice with and without tumors. Evaluation of the pharmacokinetics of hJAA-F11 constructs is critically important to the